Agents Inducing Vascularisation

The present invention relates to agents capable of inducing vascularisation. The disclosure also relates to treatment of diseases, such as cardiovascular diseases using said agents.

Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels. CVD includes coronary artery diseases (CAD) such as angina and myocardial infarction (commonly known as a heart attack). Other CVDs include stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.

The underlying mechanisms vary depending on the disease. It is estimated that dietary risk factors are associated with 53% of CVD deaths. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis. This may be caused by high blood pressure, smoking, diabetes mellitus, lack of exercise, obesity, high blood cholesterol, poor diet, excessive alcohol consumption, and poor sleep, among other things. High blood pressure is estimated to account for approximately 13% of CVD deaths, while tobacco accounts for 9%, diabetes 6%, lack of exercise 6%, and obesity 5%. Rheumatic heart disease may follow untreated strep throat.

The inventors of the present disclosure have identified peptides capable of inducing vascularisation. Thus, said peptides are promising candidates for the treatment of various diseases or disorders associated with reduced angiogenesis, including cardiovascular diseases.

One aspect of the disclosure provides for an agent comprising:

In one aspect, the present invention relates to a method for treating or preventing a disease or disorder in a subject, wherein the disease or disorder is selected from the group consisting of:

In one aspect, the present invention relates to use of an agent as defined herein for the manufacture of a medicament for the treatment or prevention of a disease or disorder selected from the group consisting of:

In one aspect, the present invention relates to an agent as defined herein for use in improving vascularisation post surgery in a subject.

In one aspect, the present invention relates to a method for inducing vascularization, said method comprising administering the agent as defined herein to a subject.

In one aspect, the present invention relates to an implant comprising a peptide as defined herein.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.

The term “some embodiments” can include one, or more than one embodiment.

The use of the word “a” or “an” when used throughout the text or in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” “Preventing” or “Prevention” as used herein, includes delaying, stopping, reducing the risk of the onset, of disease, disorder, or condition.

The agent of the present invention may be a peptide; a polynucleotide encoding said peptide; a vector comprising said polynucleotide; or a cell comprising said polynucleotide or vector.

In one embodiment, the agent is a peptide or a pharmaceutically acceptable salt thereof.

The term ‘amino acid’ as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the ‘D’ form (as compared to the natural ‘L’ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., α,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatized amino acids (see below).

When an amino acid is being specifically enumerated, such as ‘alanine’ or ‘Ala’ or ‘A’, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for peptides of the present disclosure, as long as the desired functional property is retained by the peptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.

In one embodiment, the peptide is non-naturally occurring, such as a peptide comprising non-proteinogenic amino acid residues.

In one embodiment, the agent comprises or consists of a tandem repeat comprising two or more repeat units. In one embodiment, the repeat unit comprises or consists of the amino acid sequence of any one or more of the sequences as described herein.

In one embodiment, the peptide is cyclic. The cyclic structure may be achieved by any suitable method of synthesis. Thus, heterodetic linkages may include, but are not limited to formation via disulphide, cysteine, alkylene or sulphide bridges. In one embodiment, the peptide is capable of forming at least one intramolecular cysteine bridge.

In one embodiment, the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:

In one embodiment, the peptide comprises or consisted of the amino acid sequence IELSYGIK (SEQ ID NO: 109).

In one embodiment, the peptide comprises or consists of VDTYDGGISVVYGLR (SEQ ID NO: 6). In one embodiment, the peptide comprises or consists of AEIDSIELSYGIK (SEQ ID NO: 110). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYGLR (SEQ ID NO: 7). In one embodiment, the peptide comprises or consists of DTYDGDISVVYGLR (SEQ ID NO: 8). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLRS (SEQ ID NO: 9). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLR (SEQ ID NO: 10). In one embodiment, the peptide comprises or consists of YDGDISWYGLRS (SEQ ID NO: 11). In one embodiment, the peptide comprises or consists of YDGDISVVYGLR (SEQ ID NO: 12). In one embodiment, the peptide comprises or consists of DGDISVVYGLRS (SEQ ID NO: 13). In one embodiment, the peptide comprises or consists of DGDISVVYGLR (SEQ ID NO: 14). In one embodiment, the peptide comprises or consists of GDISVVYGLRS (SEQ ID NO: 15). In one embodiment, the peptide comprises or consists of GDISVVYGLR (SEQ ID NO: 16). In one embodiment, the peptide comprises or consists of DISVVYGLRS (SEQ ID NO: 17). In one embodiment, the peptide comprises or consists of DISVVYGLR (SEQ ID NO: 18). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGLR (SEQ ID NO: 19). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLRS (SEQ ID NO: 20). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLR (SEQ ID NO: 21). In one embodiment, the peptide comprises or consists of VPNGDISLAYGLRS (SEQ ID NO: 22). In one embodiment, the peptide comprises or consists of VPNGDISLAYGLR (SEQ ID NO: 23). In one embodiment, the peptide comprises or consists of PNGDISLAYGLRS (SEQ ID NO: 24). In one embodiment, the peptide comprises or consists of PNGDISLAYGLR (SEQ ID NO: 25). In one embodiment, the peptide comprises or consists of NGDISLAYGLRS (SEQ ID NO: 26). In one embodiment, the peptide comprises or consists of NGDISLAYGLR (SEQ ID NO: 27). In one embodiment, the peptide comprises or consists of GDISLAYGLRS (SEQ ID NO: 28). In one embodiment, the peptide comprises or consists of GDISLAYGLR (SEQ ID NO: 29). In one embodiment, the peptide comprises or consists of DISLAYGLRS (SEQ ID NO: 30). In one embodiment, the peptide comprises or consists of DISLAYGLR (SEQ ID NO: 31). In one embodiment, the peptide comprises or consists of VDTYDGDGSVVYGLR (SEQ ID NO: 32). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYGLR (SEQ ID NO: 33). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYGIK (SEQ ID NO: 111). In one embodiment, the peptide comprises or consists of KCLAECDSIELSYGIK (Cyclic) (SEQ ID NO: 112). In one embodiment, the peptide comprises or consists of KPLAEDISIELSYGIK (SEQ ID NO: 113). In one embodiment, the peptide comprises or consists of KPLAEISDIELSYGIK (SEQ ID NO: 114). In one embodiment, the peptide comprises or consists of KPLAEIGDIELSYGIK (SEQ ID NO: 115). In one embodiment, the peptide comprises or consists of KPLAEGDIELSYGIK (SEQ ID NO: 116). In one embodiment, the peptide comprises or consists of KPLAEIELSYGIK (SEQ ID NO: 117). In one embodiment, the peptide comprises or consists of KPLAEIDSIELTYGIK (SEQ ID NO: 118). In one embodiment, the peptide comprises or consists of KPLAEIDGIELSYGIK (SEQ ID NO: 119). In one embodiment, the peptide comprises or consists of KPLAEIDGIELTYGIK (SEQ ID NO: 120). In one embodiment, the peptide comprises or consists of KPLAEIGSIELSYGIK (SEQ ID NO: 121). In one embodiment, the peptide comprises or consists of KGLAEIDSIELSYGIK (SEQ ID NO: 122). In one embodiment, the peptide comprises or consists of KPLAGIDSIGLSYGIK (SEQ ID NO: 123). In one embodiment, the peptide comprises or consists of Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124). In one embodiment, the peptide comprises or consists of LAEIDSIELSYGIK (SEQ ID NO: 125). In one embodiment, the peptide comprises or consists of EIDSIELSYGIK (SEQ ID NO: 126).

In one embodiment, the peptide comprises or consists of IDSIELSYGIK (SEQ ID NO: 127). In one embodiment, the peptide comprises or consists of DSIELSYGIK (SEQ ID NO: 128). In one embodiment, the peptide comprises or consists of SIELSYGIK (SEQ ID NO: 129). In one embodiment, the peptide comprises or consists of IELSYGIK (SEQ ID NO: 109). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYGL (SEQ ID NO: 34). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYG (SEQ ID NO: 35). In one embodiment, the peptide comprises or consists of VDTYDGDISVVY (SEQ ID NO: 36). In one embodiment, the peptide comprises or consists of VDTYDGDISVV (SEQ ID NO: 37). In one embodiment, the peptide comprises or consists of VDTYDGDISV (SEQ ID NO: 38). In one embodiment, the peptide comprises or consists of VDTYDGDIS (SEQ ID NO: 39). In one embodiment, the peptide comprises or consists of VDTYDGRGDSVVYGLR (SEQ ID NO: 40). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGL (SEQ ID NO: 41). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYG (SEQ ID NO: 42). In one embodiment, the peptide comprises or consists of VDVPNGDISLA (SEQ ID NO: 43). In one embodiment, the peptide comprises or consists of VDVPNGDIS (SEQ ID NO: 44). In one embodiment, the peptide comprises or consists of GDPNDGRGDSVVYGLR (SEQ ID NO: 45 In one embodiment, the peptide comprises or consists of LDGLVRAYDNISPVG (SEQ ID NO: 46). In one embodiment, the peptide comprises or consists of GDPNGDISVVYGLR (SEQ ID NO: 47). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYRLR (SEQ ID NO: 48). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYRLR (SEQ ID NO: 49). In one embodiment, the peptide comprises or consists of V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50). In one embodiment, the peptide comprises or consists of VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51 In one embodiment, the peptide comprises or consists of VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52). In one embodiment, the peptide comprises or consists of CLAEIDSC (Cyclic) (SEQ ID NO: 130). In one embodiment, the peptide comprises or consists of CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131). In one embodiment, the peptide comprises or consists of Cyclic CFKPLAEIDSIEC (SEQ ID NO: 132). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYGI (SEQ ID NO: 133). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYG (SEQ ID NO: 134). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136). In one embodiment, the peptide comprises or consists of KPLAEIDSIEL (SEQ ID NO: 137). In one embodiment, the peptide comprises or consists of KPLAEIDSIE (SEQ ID NO: 138).

The agent as defined herein can be in the form of a pharmaceutically acceptable salt or prodrug of said agent. In one embodiment, the agent as defined herein can be formulated as a pharmaceutically acceptable addition salt or hydrate of said agent, such as but not limited to K, Na, as well as non-salt e.g. H.

In one embodiment, the agent is a chemical derivative of a peptide. Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatives include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.

In one embodiment, the agent is further modified such by glycosylation, PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

In some embodiments, the agent is further conjugated to a moiety, which may be selected from the group consisting of polyethylene glycol (PEG), monosaccharides, fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides. In one embodiment, the fluorophore is selected from the group consisting of Lucifer yellow, biotin, 5,6-carboxyltetramethylrhodamine (TAMRA), indodicarbocyanine (C5) Alexa Fluor®488, Alexa Fluor®532, Alexa Fluor®647, ATTO 488, ATTO 532, 6-carboxyfluorescein (6-FAM), Alexa Fluor®350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5′-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachloro-fluorescein, Alexa Fluor®555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7.5), ATTO 550, Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY-649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700, IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm, CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm, Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes.). In one embodiment, the agent further comprises a detectable moiety, such as a moiety that is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging. In one embodiment, the detectable moiety comprises or consists of a radioisotope, such as selected from the group consisting ofTc,In,Ga,Ga,As,Zr,I andTl.

In one embodiment, the peptide comprises or consists of a fusion. For example, the peptide may comprise a fusion of two amino acid sequences as disclosed herein.

The term ‘fusion’ of a peptide relates to an amino acid sequence fused to another peptide. For example, the said peptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said peptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said peptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said peptide are also included in the scope of the disclosure. Alternatively, the fused portion may be a lipophilic molecule or peptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.